Formulation of Curcuminoids with Enhanced Bioavailability of Curcumin, Demethoxycurcumin,  Bisdemethoxycurcumin and method of preparation and uses thereof

ABSTRACT

A formulation of curcuminoid mixture having curcumin, demethoxycurcumin and bisdemethoxycurcumin and an essential oil of turmeric having 45% Ar-turmerone. The formulation gives enhanced bioavailability of bisdemethoxycurcumin, demethoxycurcumin and curcumin for up to 10 hrs. The curcuminoid mixture has curcumin, demethoxycurcumin and bisdemethoxycurcumin in a weight/weight ratio ranging from about 0.8:1:1 to about 1:1.1:1.2. A process of preparing a formulation of curcuminoid mixture having curcumin, demethoxycurcumin and bisdemethoxycurcumin in 0.8:1:1 to 1:1.1:1.2 weight/weight ratio and an essential oil of turmeric having 45% Ar-turmerone. A formulation for improved biological activity and bioavailability and tissue distribution of curcumin, demethoxycurcumin and bisdemethoxycurcumin.

This Application is a continuation of co-pending U.S. patent applicationSer. No. 15/288,882, filed Oct. 7, 2016, which is a divisional of U.S.patent application Ser. No. 14/206,044, filed Mar. 12, 2014, which is anonprovisional of U.S. Provisional Application 61/794,175 filed Mar. 15,2013 and a continuation-in-part of U.S. application Ser. No. 13/645,031filed Oct. 4, 2012, which is a continuation in part of PCT ApplicationSerial No. PCT/IN2011/000232, filed Apr. 4, 2011, which claims priorityof Indian Provisional Application Serial No. 950/CHE/2010, filed Apr. 5,2010, and a continuation-in-part of co-pending U.S. application Ser. No.14/094,725, filed Dec. 2, 2013, which is a divisional of Ser. No.13/385,717, filed Mar. 5, 2012, which is a divisional of Ser. No.12/926,985 filed Dec. 21, 2010, which is a divisional of Ser. No.12/662,740 filed Apr. 30, 2010, which is a divisional of U.S.application Ser. No. 11/635,599 filed Dec. 8, 2006, which is acontinuation of PCT Application Serial No. PCT/IN05/00176, filed May 30,2005, and a continuation-in-part of co-pending U.S. application Ser. No.13/674,249, filed Nov. 12, 2012, which is a divisional of U.S.application Ser. No. 13/506,572, filed Apr. 30, 2012, which is adivisional of Ser. No. 12/926,980, filed Dec. 21, 2010, which is adivisional of Ser. No. 12/073,864, filed Mar. 11, 2008, which is acontinuation-in-part of Ser. No. 11/635,599, filed Dec. 8, 2006, whichis a continuation of PCT Application Serial No. PCT/IN05/00176, filedMay 30, 2005, all of which applications are incorporated in entirety byreference. This application is a continuation-in-part of the followingapplications: U.S. application Ser. No. 14/476,555 filed Sep. 3, 2014,U.S. application Ser. No. 14/450,292, filed Oct. 21, 2014, U.S.application Ser. No. 14/623,608, filed Feb. 17, 2015, U.S. applicationSer. No. 14/698,944, filed Apr. 29, 2015, and, U.S. application Ser. No.14/800,950, filed Jul. 16, 2015, all of which applications areincorporated in entirety by reference.

FIELD OF INVENTION

Disclosure relates to a formulation of curcuminoid mixture and essentialoil of turmeric having 45% Ar-turmerone. The formulation gives enhancedbioavailability of bisdemethoxycurcumin, demethoxycurcumin in additionto curcumin for up to 10 hrs. The curcuminoid mixture comprisescurcumin, demethoxycurcumin and bisdemethoxycurcumin in which curcuminranges from about 0.8 to about 1.0 part weight/weight, demethoxycurcumin ranges from about 1.0 to about 1.1 part weight/weight andbisdemethoxy curcumin ranges from about 1.0 to about 1.2 partweight/weight. In the curcuminoid mixture the ratio of curcumin,demethoxy curcumin and bisdemethoxy curcumin ranges from about 0.8:1:1to about 1:1.1:1.2 weight/weight ratio. The formulation providesimproved biological activity and bioavailability of curcumin,demthoxycurcumin and bisdemethoxycurcumin.

BACKGROUND OF THE INVENTION

Turmeric is a spice grown in India and other tropical regions of Asia.It has a long history of use in herbal remedies, particularly in China,India, and Indonesia. Turmeric or “yellow root” is a general term forplants and plant materials having a high content of curcuminoids,compounds that have a strong colouring effect and which are usedextensively in the colouring of e.g. food products.

Curcumin, reddish orange and with two methoxy groups, is the principalcurcuminoid of the popular Indian spice turmeric, which is a member ofthe ginger family (Zingiberaceae). Turmeric's other two curcuminoids aredemethoxycurcumin, orange-yellow with one methoxy group, andbisdemethoxycurcumin, yellow and without a methoxy group. Thecurcuminoids are natural phenols that are responsible for the yellowcolor of turmeric.

Preparing solvent extracts of Curcuma plant materials, in particularrhizomes, provides an oleoresin comprising an essential oil of turmericand curcuminoids. The oleoresin may subsequently be subjected to acrystallization step resulting in the obtainment of curcuminoid crystalsof a relatively high purity and a liquid part. Liquid part includedessential oil of turmeric, flavouring compounds, any impurities thatremained in solution and, curcuminoids that did not crystallize.Essential oil of turmeric is isolated from the liquid part by steamdistillation. The residual material that remains after the aboveseparation of curcuminoid crystals and essential oil of turmericconsists mainly of uncrystallized curcuminoids. This residue isgenerally disposed of as a waste product of the industrial process.

SUMMARY

The disclosure provides a composition for enhanced bioavailability ofcurcumin, demethoxycurcumin and bisdemethoxycurcumin. The compositionincludes a curcuminoid mixture and essential oil of turmeric. Thecurcuminoid mixture includes curcumin, demethoxycurcumin andbisdemethoxycurcumin in a weight ratio ofcurcumin:demethoxycurcumin:bisdemethoxycurcumin of about 0.8:1:1 toabout 1:1.1:1.2. The essential oil of turmeric includes about 45%Ar-turmerone. The weight ratio of the curcuminoid mixture to essentialoil of turmeric ranges from about 1:3 to about 99:1. In some embodimentsthe weight ratio of the curcuminoid mixture to essential oil of turmericranges is about 10:1. In some embodiments the weight ratio of thecurcuminoid mixture to essential oil of turmeric ranges is about 12:1.

The disclosure provides a formulation which gives enhancedbioavailability of bisdemethoxycurcumin, demethoxycurcumin, and curcuminfor up to 10 hrs.

Disclosure provides a process for obtaining curcuminoid mixture withcurcumin, demethoxycurcumin and bisdemethoxycurcumin blended withessential oil of turmeric having 45% Ar-turmerone. More specifically,the disclosure provides a process wherein the residue, previouslyconsidered as a waste obtained after the separation of essential oil andcurcuminoids from oleoresin, is made useful by a silica impregnationmethod. The process results a curcuminoid mixture having curcumin,demethoxycurcumin and bisdemethoxycurcumin in 0.8:1:1 to 1:1.1:1.2weight/weight ratio.

In some embodiments, the ratio of the three curcuminoid mixture obtainedfrom waste obtained after the separation of essential oil andcurcuminoids from oleoresin is about 10-90% of curcumin, about 6-70% ofdemethoxycurcumin and about 2-60% of bisdemethoxycurcumin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides Table 1. Table 1 shows percentage inhibition ofinflammation in carrageenan induced paw oedema model.

FIG. 2 provides Table 5. Table 5 provides bioavailability of curcumin,demethoxycurcumin and bisdemethoxycurcumin in human subjects followingby administration of different extracts.

DETAILED DESCRIPTION

The disclosure provides a process wherein the residue, previouslyconsidered as a waste obtained after the separation of essential oil andcurcuminoids from oleoresin, is made useful by a silica impregnationmethod. The process results a curcuminoid mixture having curcumin,demethoxycurcumin and bisdemethoxycurcumin in about 0.8:1:1 to about1:1.1:1.2 weight/weight ratio. The disclosure provides a formulation ofcurcuminoid mixture having curcumin, demethoxycurcumin andbisdemethoxycurcumin in 0.8:1:1 to 1:1.1:1.2 weight/weight ratio and anessential oil of turmeric having 45% Ar-turmerone. The formulation givesenhanced bioavailability of bisdemethoxycurcumin, demethoxycurcumin inaddition to curcumin for up to 10 hrs.

In some embodiments, the formulation provides a curcuminoid mixturehaving curcumin, demethoxycurcumin and bisdemethoxycurcumin ranging fromabout 0.8:1:1 to about 1:1.1:1.2 of a weight/weight ratio ofcurcumin:demethoxycurcumin:bisdemethoxycurcumin. This range of weightratio of the curcumin, demethoxycurcumin and bisdemethoxy curcumin isobtained by extraction of the curcumin from a turmeric waste residueobtained after the extraction of curcuminoid crystals from turmericrhizomes. The turmeric waste residue is obtained after the separation ofessential oil and curcuminoid crystals from turmeric oleoresin.Curcuminoid mixture having curcumin, demethoxycurcumin andbisdemethoxycurcumin in 0.8:1:1 to 1:1.1:1.2 weight/weight ratio whenblended with essential oil of turmeric having 45% Ar-turmerone gives aformulation having improved biological activity and bioavailability ofcurcumin, demethoxycurcumin and bisdemethoxycurcumin, as compared tobiological activity and bioavailability of a formulation havingcurcuminoid crystals alone, or a formulation having curcuminoid crystalsblended with 45% Ar-turmerone, or a formulation of curcuminoid mixturealone having a weight ratio ofcurcumin:demethoxycurcumin:bisdemethoxycurcumin ranging from about0.8:1:1.

In one embodiment, the ratio of the three curcuminoid mixture obtainedfrom waste after the separation of essential oil and curcuminoids fromoleoresin is typically: about 10-90% curcumin, about 6-70%demethoxycurcumin and about 2-60% bisdemethoxycurcumin.

The volatile oil of turmeric was isolated by conventional methods ofsteam distillation to isolate essential oils and is well known in theart.

Curcuminoids are isolated from waste or residue material. The waste orresidue material is obtained after the separation of essential oil andcurcuminoids from oleoresin, by silica impregnation method. The waste orresidue material is passed through a silica column. The silica column iswashed with methanol to obtain three methanol fractions. Methanolfractions 2 and 3 have a mixture of curcuminoids. The combination ofmethanol fractions 2 and 3 provides a curcuminoid mixture havingcurcumin, demethoxycurcumin and bisdemethoxycurcumin in a 0.8:1:1 to1:1.1:1.2 weight/weight ratio ofcurcumin:demethoxycurcumin:bisdemethoxycurcumin.

Curcuminoid mixture and volatile oils of turmeric having 45%Ar-turmerone are mixed and blended to get a uniform product. The producthas curcuminoid mixture in 0.8:1:1 to 1:1.1:1.2 weight/weight ratio ofcurcumin to demethoxycurcumin to bisdemethoxycurcumin.

The disclosure provides a method of extracting curcuminoid mixture in0.8:1:1 to 1:1.1:1.2 weight/weight ratio ofcurcumin:demethoxycurcumin:bisdemethoxycurcumin from residue or wasteafter the separation of essential oil and curcuminoids from oleoresin ofturmeric. The disclosure provides a method of extracting curcuminoidmixture from residue or waste after the separation of essential oil andcurcuminoid crystals from oleoresin of turmeric. The disclosure providesa concentrated solution prepared by solvent treatment of dried turmericrhizomes. This concentrated solution is also referred to as oleoresin.Curcuminoids are crystallized from the oleoresin, leaving behind a wasteliquid which contains essential oil. It is found that the waste liquidcontains essential oil also contains curcuminoids, however in industrialprocess the liquid can be referred to as “waste” as far as curcuminoidextraction is concerned because the liquid is discarded with no furtherextraction of curcuminoids is performed from this liquid. The disclosureprovides a method to extract essential oil of turmeric from this liquidindustrial waste. The disclosure provides a method for extracting thecurcuminoids from this liquid industrial waste. The disclosure providesa method of steam distilling essential oils from the waste liquid. Aftersteam distillation of essential oil from the waste liquid, a residue isobtained. This waste residue is discarded as industrial waste. Thedisclosure provides a method to extract curcuminoids having aweight/weight ratio of curcumin:demethoxycurcumin:bisdemethoxycurcuminranging from about 0.8:1:1 to about 1:1.1:1.2 from the waste residue.

In some embodiments of the method of preparing curcuminoid mixturehaving a weight/weight ratio ofcurcumin:demethoxycurcumin:bisdemethoxycurcumin ranging from about0.8:1:1 to about 1:1.1:1.2, rhizomes of turmeric are dried to form driedturmeric rhizomes. The dried turmeric rhizomes are powdered to form adried turmeric rhizome powder. Ethyl acetate is added to the driedturmeric rhizome powder to obtain an ethyl acetate extract solution anda residue. The residue is separated from the ethyl acetate extractsolution. The ethyl acetate extract solution is filtered. The solvent isstripped from the filtered ethyl acetate extract solution to form anethyl acetate extract. The ethyl acetate extract is cooled to obtaincrystals of curcuminoid and a liquid. The liquid includes essential oilof turmeric and curcuminoids that did not crystallize. The liquid issteam distilled to obtain essential oil of turmeric and a waste residue(also referred to as waste residue 2). The waste residue 2 is loadedonto a silica column. The silica column is washed with methanol. Threefractions of methanol eluate are collected and referred to as Fraction1, Fraction 2 and Fraction 3. Fraction 2 and Fraction 3 are combined toobtain a combined methanol fraction. The combined methanol fraction isconcentrated under heat and vacuum to obtain a concentrated methanolfraction. The concentrated methanol fraction is dried to obtain a driedmethanol extract. The dried methanol extract is powdered to obtain apowder having a curcuminoid mixture of curcumin, demethoxycurcumin andbisdemethoxycurcumin in a weight ratio ofcurcumin:demethoxycurcumin:bisdemethoxycurcumin of about 0.8:1:1 toabout 1:1.1:1.2. In some embodiments of the method, other solventsincluding acetone, hexane, ethyl acetate and combinations thereof can beused instead of only the ethyl acetate solvent described above.

The essential oil is fractionated to obtain three fractions. One of thefractions contains essential oil of turmeric having 45% Ar-turmerone.

The disclosure provides a method of preparing a formulation ofcurcuminoids in 0.8:1:1 to 1:1.1:1.2 weight/weight ratio ofcurcumin:demethoxycurcumin:bisdemethoxycurcumin and essential oil ofturmeric having 45% Ar-turmerone. The curcuminoid mixture of curcumin,demethoxycurcumin and bisdemethoxycurcumin in a weight ratio ofcurcumin:demethoxycurcumin:bisdemethoxycurcumin of about 0.8:1:1 toabout 1:1.1:1.2, is suspended in water to form a suspension. Essentialoil having 45% Ar-turmerone is added to the suspension to form amixture. The mixture is homogenized to form slurry. The slurry is driedunder heat and vacuum to form a uniform blend of formulation. Theformulation has curcuminoid mixture in 0.8:1:1 to 1:1.1:1.2weight/weight ratio of curcumin:demethoxycurcumin:bisdemethoxycurcuminand an essential oil of turmeric having 45% Ar-turmerone. The slurry canbe dried under heat and vacuum using, for example, a vaccumizeddesolventiser having a stirrer.

In one embodiment, a homogeneous mixture of curcuminoid mixture andwater is prepared by suspending the curcuminoid mixture in about 3 to 5times its quantity of water to form a suspension. The suspension ishomogenized to obtain slurry. The slurry is dried under heat and vacuumto form a formulation having a homogeneous mixture of the curcuminoidmixture and water.

The disclosure provides a dosage form of the formulation of curcuminoidsin 0.8:1:1 to 1:1.1:1.2 weight/weight ratio ofcurcumin:demethoxycurcumin:bisdemethoxycurcumin and essential oil ofturmeric having 45% Ar-turmerone. The disclosure provides a dosage formof the formulation of curcuminoids in 0.8:1:1 to 1:1.1:1.2 weight/weightratio of curcumin:demethoxycurcumin:bisdemethoxycurcumin and essentialoil of turmeric having 45% Ar-turmerone for oral administration. Dosageforms of the formulation are selected from the group consisting of thehard gelatin capsule, soft gelatin capsule, tablet, sachet, powder,paste, solution, suspension, emulsion, pills etc.

The disclosure provides a method of preparing a gelatin capsule havingcurcuminoids in 0.8:1:1 to 1:1.1:1.2 weight/weight ratio ofcurcumin:demethoxycurcumin:bisdemethoxycurcumin and essential oil ofturmeric having 45% Ar-turmerone by suspending curcuminoid mixture inwater to form a suspension. Then essential oil turmeric is added to thesuspension to form a mixture. Then the mixture is homogenized to obtaina slurry. Then the slurry is dried under heat and vacuum to form auniform blend of a composition having the curcuminoid and the essentialoil of turmeric. Then the blend is filled into a gelatin capsule.Gelatin capsules are prepared by a capsule filling machine manufacturedby Pam Pharmaceuticals, Mumbai, India.

In some embodiments, the weight ratio of the curcuminoid mixture, whichhas a 0.8:1:1 to 1:1.1:1.2 weight/weight ratio ofcurcumin:demethoxycurcumin:bisdemethoxycurcumin, to the essential oil ofturmeric ranges from about 1:1 to about 90:1. The curcuminoid mixturehaving curcumin:demethoxycurcumin:bisdemethoxycurcumin in a 0.8:1:1 to1:1.1:1.2 weight/weight ratio is also referred to as curcuminoidmixture. In some embodiments, the weight ratio of the curcuminoidmixture to the essential oil of turmeric ranges from about 1:1 to about3:1. The weight ratio of the curcuminoid mixture to the essential oil ofturmeric can be varied from about 3:1 to about 99:1. In someembodiments, the weight ratio of the curcuminoid mixture to theessential oil of turmeric ranges from about 1:1 to about 70:1. In someembodiments, the weight ratio of the curcuminoid mixture to theessential oil of turmeric ranges from about 1:1 to about 45:1. In someembodiments, the weight ratio of the curcuminoid mixture to theessential oil of turmeric ranges from about 3:1 to about 50:1. In someembodiments, the weight ratio of the curcuminoid mixture to theessential oil of turmeric ranges from about 8:1 to about 25:1. In someembodiments, the weight ratio of the curcuminoid mixture to theessential oil of turmeric is about 90:7. In some embodiments, the weightratio of the curcuminoid mixture to the essential oil of turmeric isabout 90:8. In some embodiments, the weight ratio of the curcuminoidmixture to the essential oil of turmeric is about 90:9. In someembodiments, the weight ratio of the curcuminoid mixture to theessential oil of turmeric is about 89:9. In some embodiments, the weightratio of the curcuminoid mixture to the essential oil of turmeric isabout 89:8. In one embodiment, the ratio is about 85:15. In anotherembodiment, the ratio is about 92:8. In another embodiment, the ratio isabout 95:5. In another embodiment the weight ratio is about 10:1. Insome embodiments, the weight ratio is about 12:1. In some embodiments,the weight ratio of the curcuminoid mixture to the essential oil ofturmeric is about 1:2. In some embodiments, the weight ratio of thecurcuminoid mixture to the essential oil of turmeric is about 2:1. Insome embodiments, the weight ratio of the curcuminoid mixture to theessential oil of turmeric ranges from about 1:3 to about 99:1.

Some embodiments provide a method of enhancing bioavailability ofcurcumin by administering the formulation of curcuminoid mixture andessential oil of turmeric. Some embodiments provide a method ofenhancing bioavailability of demethoxycurcumin by administering thecomposition of curcuminoid mixture and essential oil of turmeric. Someembodiments provide a method of enhancing bioavailability ofbisdemethoxycurcumin by administering the composition of curcuminoidmixture and essential oil of turmeric. Some embodiments provide a methodof enhancing bioavailability of curcumin, demethoxy curcumin andbisdemethoxy curcumin in a tissue by administering the composition ofcurcuminoid mixture and essential oil of turmeric. Enhancement ofbioavailability of curcumin is observed in tissues including lungs,heart, kidney, brain, liver, pancreas, stomach, intestine and skin.Enhancement of bioavailability of demethoxy curcumin is observed intissues including lungs, heart, kidney, brain, liver, pancreas, stomach,intestine and skin. Enhancement of bioavailability of bisdemethoxycurcumin is observed in tissues including lungs, heart, kidney, brain,liver, pancreas, stomach, intestine and skin.

Another embodiment of the present invention provides a formulationhaving a curcuminoid mixture in 0.9:1:1.1 weight/weight ratio ofcurcumin:demethoxycurcumin:bisdemethoxycurcumin and a ratio ofcurcuminoid mixture: essential oil of turmeric of about 10:1 or about12:1 ratio. The essential oil of turmeric includes 45% Ar-turmerone. Theformulation has anti-inflammatory activity.

Inflammation is a complex biological response of vascular tissues andimmune cells to harmful stimuli, such as pathogens, damaged cells, orirritants. It is characterized by five signs: redness, increased heat,swelling, pain, and/or loss of function. Inflammation can be classifiedas either acute or chronic. Acute inflammation is the initial responseof the body to harmful stimuli and is achieved by the increased movementof plasma and leukocytes (especially granulocytes) from the blood intothe injured tissues. A cascade of biochemical events propagates andmatures the inflammatory response, involving the local vascular system,the immune system, and various cells within the injured tissue.Prolonged inflammation, known as chronic inflammation, leads to aprogressive shift in the type of cells present at the site ofinflammation and is characterized by simultaneous destruction andhealing of the tissue from the inflammatory process. At present, themajority of medicines widely used as anti-inflammatory agents arenonsteroid anti-inflammatory drugs (NSAIDs) that have, as the mechanismof action, an inhibitory action on cyclooxygenases (COXs) that isinvolved in the biosynthesis of prostanoids. However, since prostanoidsynthesis activity is present in various tissues in the living body andgoverns the homeostasis thereof, various side effects are induced whenNSAID is administered. One test for inflammation is the C-reactiveprotein (CRP) measurement. Some clinicians are advocating including itroutinely. The CRP test detects any inflammation, no matter where it is.A skinned knee, flu, arthritis and infections are common causes ofelevated CRP. Anti-inflammatory activity of extracts/drugs can beevaluated in small animals like rats and mice. A classic model iscarrageenan induced paw oedema model in rats. Carrageenan is an irritantand produces inflammation in rats paw after injecting a small volumeinto sub-plantar region. The volume of paw increases within 30 minutesof injecting carrageenan which can be measured by a plethysmometer.Volume of paw should be measured at various time points aftercarrageenan and test drug administration and should be compared withcontrol.

An anti-inflammatory activity of formulation having a curcuminoidmixture in about a 0.9:1:1.1 weight/weight ratio ofcurcumin:demethoxycurcumin:bisdemethoxycurcumin (C:D:B) and essentialoil of turmeric having 45% Ar-turmerone in about a 10:1 and about 12:1ratios showed higher percentage inhibition of inflammation. A highervalue of percentage inhibition indicates more anti-inflammatoryactivity. Whereas curcuminoid mixture having C:D:B in 0.9:1:1.1 w/wratio and regular turmeric extract showed negative percentage inhibitionof inflammation. A negative percentage inhibition indicates that testmaterial did not showing anti-inflammatory effect. Formulation ofcurcuminoid mixture having C:D:B in 0.9:1:1.1 ratio blended withessential oil of turmeric having 45% Ar-turmerone in 10:1 or in 12:1ratios shows a decrease in inflammation from about 30 mins to about 24hours.

Some embodiments provide a composition to decrease inflammation. Thecomposition includes a curcuminoid mixture and essential oil ofturmeric. The curcuminoid mixture includes curcumin, demethoxycurcuminand bisdemethoxycurcumin in a weight ratio ofcurcumin:demethoxycurcumin:bisdemethoxycurcumin of about 0.8:1:1 toabout 1:1.1:1.2. The essential oil of turmeric includes about 45%Ar-turmerone. Some embodiments provide a method to decrease inflammationby administering the composition of curcuminoid mixture and essentialoil of turmeric. In some embodiments, the decrease in inflammation isobserved from about 30 mins to about 24 hours following administrationof the composition of curcuminoid mixture and essential oil of turmeric.

Some embodiments provide a composition to decrease swelling. Thecomposition includes a curcuminoid mixture and an essential oil ofturmeric. The curcuminoid mixture includes curcumin, demethoxycurcuminand bisdemethoxycurcumin in a weight ratio ofcurcumin:demethoxycurcumin:bisdemethoxycurcumin of about 0.8:1:1 toabout 1:1.1:1.2. The essential oil of turmeric includes about 45%Ar-turmerone. Some embodiments provide a method of decreasing swellingby administering the composition of curcuminoid mixture and essentialoil of turmeric.

In some embodiments of the formulation having curcuminoid mixture havingC:D:B in 0.9:1:1.1 ratio blended with essential oil of turmeric having45% Ar-turmerone in 10:1 or in 12:1 ratios, enhances bioavailability ofcurcumin, demethoxycurcumin and bisdemethoxycurcumin in the bodytissues.

In another embodiment, the formulation of curcuminoid mixture havingC:D:B in 0.9:1:1.1 ratio blended with essential oil of turmeric having45% Ar-turmerone in 10:1 or in 12:1 ratios, enhances the bioavailabilityof curcumin, demethoxycurcumin and bisdemethoxycurcumin in the blood forup to 10 hrs. After administering capsules having a 10:1 weight ratio ofcurcuminoid mixture having C:D:B in 0.9:1:1.1 w/w ratio blended withessential oil of turmeric having 45% Ar-turmerone, the maximumabsorption for curcumin was 102.6 ng/gm, for demethoxycurcumin 120.2ng/gm and for bisdemethoxycurcumin 140.7 ng/gm. After administeringcapsules having a 12:1 weight ratio of curcuminoid mixture having C:D:Bin 0.9:1:1.1 w/w ratio blended with essential oil of turmeric having 45%Ar-turmerone, the maximum absorption for curcumin was 118.1 ng/gm, fordemethoxycurcumin 125.1 ng/gm and for bisdemethoxycurcumin 152.5 ng/gm.Treating subjects with curcuminoid mixture having C:D:B in 0.9:1:1.1 w/wratio shows low detection of curcumin, demethoxycurcumin andbisdemethoxycurcumin.

EXAMPLES Example 1 Method of Preparation of Regular Turmeric Extract

The rhizomes of turmeric (500 Kg) were dried. The dried turmericrhizomes were powdered to form powdered turmeric. Ethyl acetateextraction of the powdered turmeric was performed. For the extraction,ethyl acetate (1500 L) was added to the powdered turmeric at 78° C.temperature for 1 hr. After ethyl acetate extraction a solution and aresidue 1 were obtained. The residue 1 was separated from the solutionand 1500 liters of ethyl acetate was again added to the residue 1 forextraction at 78° C. temperature for 1 hr. The resultant residue wassimilarly extracted with ethyl acetate for three more times. Thesolution from each of the ethyl acetate extraction steps was combinedand filtered. The solvent from the filtered solution was stripped toform an extract. Then the extract was cooled to about 4° C. to obtaincrystals of curcuminoid (20 Kg) and a liquid. The crystals ofcurcuminoid were isolated from the liquid by filtration. The crystal ofcurcuminoid included mixture of curcumin, demethoxycurcumin andbisdemethoxycurcumin. The crystals of curcuminoid were also referred toas regular turmeric extract.

Example 2 Preparation of Fractions of Essential Oil of Turmeric

The rhizomes of turmeric (500 Kg) were dried. The dried turmericrhizomes were powdered to form powdered turmeric. Ethyl acetateextraction of the powdered turmeric was performed. For the extraction,ethyl acetate (1500 L) was added to the powdered turmeric at 78° C.temperature for 1 hr. After ethyl acetate extraction a solution and aresidue 1 were obtained. The residue 1 was separated from the solutionand 1500 liters of ethyl acetate was again added to the residue 1 forextraction at 78° C. temperature for 1 hr. The resultant residue wassimilarly extracted with ethyl acetate for three more times. Thesolution from each of the ethyl acetate extraction steps was combinedand filtered. The solvent from the filtered solution was stripped toform an extract. Then the extract was cooled to about 4° C. to obtaincrystals of curcuminoid and a liquid. The crystals of curcuminoid wereisolated from the liquid by filtration. The crystal of curcuminoidincluded mixture of curcumin, demethoxycurcumin andbisdemethoxycurcumin. The crystals of curcuminoid were referred to asregular turmeric extract.

The liquid included essential oil of turmeric, flavouring compounds, anyimpurities that remained in solution, and, curcuminoids that did notcrystallize. The liquid was then steam distilled to isolate essentialoil of turmeric having 10-15% Ar-turmerone (25 Kg) and a residue 2. Theessential oil having 10-15% Ar-turmerone was fractionated at differenttemperatures to obtain three fractions. Essential oil of turmeric having45% Ar-turmerone (7.5 Kg) was obtained as fraction 3. Essential oil ofturmeric having 4-5% Ar-turmerone (8.3 Kg) was obtained as fraction 2.Essential oil of turmeric having 2-3% Ar-turmerone (9.3 Kg) was obtainedas fraction 1.

Example 3

Method of preparation of formulation having regular turmeric extract andEssential oil of turmeric (having 45% Ar-turmerone) in a 10:1 ratio.

Regular turmeric extract (2.7 Kg) prepared as per example 1 wassuspended in water (12 L) to form a suspension. From Example 2, thefraction of essential oil (fraction 3) having 45 Ar-turmerone (0.27 Kg)was added to the suspension in 10:1 ratio of regular turmericextract:essential oil of turmeric to obtain a mixture of regularturmeric extract and essential oil of turmeric. The mixture of regularturmeric extract blended with essential oil of turmeric was pulverizedin a colloidal mill to form a slurry. Water was stripped from the slurryunder heat and vacuum to form a uniform blend (3 Kg) of formulation. Theformulation included regular turmeric extract and essential oil ofturmeric (having 45% Ar-turmerone) in 10:1 ratio.

Example 4 Preparation of Curcuminoid Mixture Having Curcumin,Demethoxycurcumin, Bisdemetoxycurcumin in a Ratio of 0.9:1:1.1.

The rhizomes of turmeric (500 Kg) were dried. The dried turmericrhizomes were powdered to form powdered turmeric. Ethyl acetateextraction of the powdered turmeric was performed. For the extraction,ethyl acetate (1500 L) was added to the powdered turmeric at 78° C.temperature for 1 hr. After ethyl acetate extraction a solution and aresidue 1 were obtained. The residue 1 was separated from the solutionand 1500 liters of ethyl acetate was again added to the residue 1 forextraction at 78° C. temperature for 1 hr. The resultant residue wassimilarly extracted with ethyl acetate for three more times. Thesolution from each of the ethyl acetate extraction steps was combinedand filtered. The solvent from the filtered solution was stripped toform an extract. Then the extract was cooled to about 4° C. to obtaincrystals of curcuminoid and a liquid. The crystals of curcuminoid wereisolated from the liquid by filtration. The crystals of curcuminoidincluded mixture of curcumin, demethoxycurcumin andbisdemethoxycurcumin. The crystals of curcuminoid were referred to asregular turmeric extract.

The liquid included essential oil of turmeric, flavouring compounds, anyimpurities that remained in solution, and, curcuminoids that did notcrystallize. The liquid was then steam distilled to isolate essentialoil of turmeric having 10-15% Ar-turmerone (25 Kg) and a residue 2. Theessential oil having 10-15% Ar-turmerone was fractionated at differenttemperatures to obtain three fractions. Essential oil having 45%Ar-turmerone (7.5 Kg) was obtained as fraction 3. Essential oil ofturmeric having 4-5 Ar-turmerone (8.3 Kg) was obtained as fraction 2.Essential oil of turmeric having 2-3% Ar-turmerone (9.3 Kg) was obtainedas fraction 1.

Residue 2 was passed through silica column. The silica column washedwith methanol and three fractions were collected. The initial 30% elutewas collected as fraction 1. The next 40% elute was collected asfraction 2. The remaining was collected as fraction 3. Methanolfractions 2 and 3 were combined and concentrated at 60-70° C. undervacuum (550 mm Hg). The concentrate was finally dried in an AgitatedThin Film Drier (ATFD). Dried extract was powdered in a pulverisor toform powder of curcuminoids. The powder of curcuminoids includedcurcumin, demethoxycurcumin and bisdemethoxycurcumin (4 Kg) in a0.9:1:1.1 w/w ratio of curcumin:demethoxycurcumin:bisdemethoxycurcumin(Curcumin 18 mg, Demethoxycurcumin 20 mg and Bisdemethoxycurcumin 22mg). The powder of curcuminoids was also referred to as curcuminoidmixture having C:D:B in 0.9:1:1.1 w/w ratio.

Example 5 Method of Preparation of Formulation Having CurcuminoidMixture and Essential Oil of Turmeric (Having 45% Ar-Turmerone) in a10:1 Ratio.

From Example 4, the curcuminoid mixture (2.7 Kg) obtained from residue 2was suspended in water (12 L) to form a suspension. From Example 2, thefraction of essential oil (fraction 3) having 45% Ar-turmerone (0.27 Kg)was added to the suspension in 10:1 ratio of curcuminoidmixture:essential oil of turmeric to obtain a mixture of curcuminoidmixture and essential oil of turmeric. The mixture of curcuminoidmixture blended with essential oil of turmeric was pulverized in acolloidal mill to form a slurry. Water was stripped from the slurryunder heat and vacuum to form a uniform blend (3 Kg) of formulation. Theformulation included curcuminoid mixture (having Curcumin 16.36 mg,Demethoxycurcumin 18.18 mg and Bisdemethoxycurcumin 20 mg) and essentialoil of turmeric (having 45% Ar-turmerone) in 10:1 ratio.

Example 6 Method of Preparation of Formulation Having CurcuminoidMixture and Essential Oil of Turmeric (Having 45% Ar-Turmerone) in a12:1 Ratio.

From Example 4, the curcuminoid mixture (3.5 Kg) obtained from residue 2was suspended in water (15 L) to form a suspension. From Example 2, thefraction of essential oil (fraction 3) having 45% Ar-turmerone (0.29 Kg)was added to the suspension in 12:1 ratio of curcuminoidmixture:essential oil of turmeric to obtain a mixture of curcuminoidmixture and essential oil of turmeric. The mixture of curcuminoidmixture and essential oil of turmeric was pulverized in a colloidal millto form slurry. Water was stripped from the slurry under heat and vacuumto form a uniform blend (3.8 Kg) of formulation. The formulationincluded curcuminoid mixture (Curcumin 16.62 mg, Demethoxycurcumin 18.46mg and Bisdemethoxycurcumin 20.30 mg and essential oil of turmeric(having 45% Ar-turmerone) in 12:1 ratio.

Example 7

Method of preparation of formulation having curcuminoid mixture andEssential oil of turmeric (having 45% Ar-turmerone) in a 1:10 ratio.

From Example 4, the curcuminoid mixture (0.27 Kg) obtained from residue2 was suspended in water (12 L) to form a suspension. From Example 2,the fraction of essential oil (fraction 3) having 45% Ar-turmerone (2.7Kg) was added to the suspension in 1:10 ratio of curcuminoidmixture:essential oil of turmeric to obtain a mixture of curcuminoidmixture and essential oil of turmeric. The mixture of curcuminoidmixture and essential oil of turmeric was pulverized in a colloidal millto form a slurry. Water was stripped from the slurry under heat andvacuum to form a uniform blend (3 Kg) of formulation. The formulationincluded curcuminoid mixture (Curcumin 1.64 mg, Demethoxycurcumin 1.82mg and Bisdemethoxycurcumin 2 mg) and essential oil of turmeric (having45% Ar-turmerone) in 1:10 ratio.

Example 8

Anti-Inflammatory Activity of Curcuminoid Mixture Blended with EssentialOil of Turmeric Having 45% Ar-Turmerone

Formulation of curcuminoid mixture and essential oil of turmeric having45% Ar-turmerone were prepared in ratio of 10:1 or 12:1 ratio ofcurcuminoid mixture:essential oil of turmeric. The curcuminoid mixtureincluded curcumin, demethoxycurcumin and bisdemethoxycurcumin in a0.8:1:1 to 1:1.1:1.2 weight/weight ratio ofcurcumin:demethoxycurcumin:bisdemethoxycurcumin.

Anti-inflammatory activity of formulations having 10:1 and 12:1 ratio ofcurcuminoid mixture (C:D:B in 0.8:1:1 to 1:1.1:1.2 weight/weightratio):essential oil of turmeric having 45% Ar-turmerone were comparedwith curcuminoid mixture (C:D:B in 0.8:1:1 to 1:1.1:1.2 weight/weightratio), regular turmeric extract blended with essential oil of turmerichaving 45% Ar-turmerone in 10:1 ratio or with regular turmeric extractin Wistar rats. The dosage administered for test samples was 20 mg ofcurcuminoids/Kg body weight of the rat. Diclofenac (standardanti-inflammatory drug) administered was 10 mg Diclofenac/Kg bodyweight.

Group 1 consisted of control animals and were fed with vehicle (1% Tween80)

Group 2 consisted of animals which were given Diclofenac standard.

Dose: 10 mg Diclofenac/Kg body weight.

Group 3 animals were given regular turmeric extract having 95%curcuminoids prepared as per Example 1, of which Curcumin 15.36 mg,Demethoxycurcumin 3.42 mg and Bisdemethoxycurcumin 1.22 mg (equivalentto 20 mg curcuminoids) suspended in 1% Tween 80.

Dose: Regular turmeric extract (equivalent to 20 mg curcuminoids)suspended in 1% Tween 80/Kg body weight.

To Group 4 animals, regular turmeric extract blended with essential oilof turmeric having 45% Ar-turmerone in 10:1 ratio prepared as perExample 3 (Curcumin 13.96 mg, Demethoxycurcumin 3.11 mg andBisdemethoxycurcumin 1.11 mg) suspended in 1% Tween 80 and fed.

Dose: Regular turmeric extract blended with essential oil of turmerichaving 45% Ar-turmerone in 10:1 ratio (equivalent to 20 mg curcuminoids)suspended in 1% Tween 80/Kg body weight.

Curcuminoid mixture having C:D:B in 0.9:1:1.1 w/w ratio (prepared as perExample 4) with Curcumin 6 mg, Demethoxycurcumin 6.67 mg andBisdemethoxycurcumin 7.33 mg was suspended in 1% Tween 80 and fed toGroup 5 animals.

Dose: 20 mg Curcuminoid mixture having C:D:B in 0.9:1:1.1 w/w ratiosuspended in 1% Tween 80/Kg body weight.

To Group 6 animals, curcuminoid mixture having C:D:B in 0.9:1:1.1 w/wratio blended with essential oil of turmeric having 45% Ar-turmerone in10:1 ratio prepared as per Example 5 suspended in 1% Tween 80 and fed.

Dose: 20 mg of the formulation (Curcumin 5.45 mg, Demethoxycurcumin 6.06mg, Bisdemethoxycurcumin 6.9 mg and essential oil of turmeric having 45%Ar-turmerone) suspended in 1% Tween 80/Kg body weight.

To Group 7 animals, curcuminoid mixture having C:D:B in 0.9:1:1.1 w/wratio blended with essential oil of turmeric having 45% Ar-turmerone in12:1 ratio prepared as per Example 6 was suspended in 1% Tween 80 wasfed.

Dose: 20 mg of the formulation (Curcumin 5.54 mg, Demethoxycurcumin 6.16mg and Bisdemethoxycurcumin 6.77 mg and essential oil of turmeric having45% Ar-turmerone in 12:1 ratio) suspended in 1% Tween 80/Kg body weight.

Vehicle (1% Tween 80), formulation having 10:1 ratio of curcuminoidmixture:essential oil of turmeric (having 45% Ar-turmerone), formulationhaving 12:1 ratio of curcuminoid mixture:essential oil of turmeric(having 45% Ar-turmerone), curcuminoid mixture alone, regular turmericextract, regular turmeric extract blended with essential oil of turmerichaving 45% Ar-turmerone in 10:1 ratio or diclofenac (a well knownanti-inflammatory drug) were administered orally to respective groups ofrats orally. Then after 30 mins, the treated rats were challenged withcarrageenan (0.1 ml, 1% carrageenan suspension in 0.9% NaCl solution) byinjecting to the animals in subplantar region of the hind paw.Carrageenan is a chemical known to induce inflammation in rats. The pawvolume was determined for all the animals at different time intervals(base line, 1 hr, 3 hr, 6 Hr, 8 hr and 24 hr) after carrageenaninjection using digital plethysmometer and the percentage inhibition ofinflammation was calculated using following formula:

% inhibition=(1−D/C)×100

D—Represents the difference in paw volume from baseline in test/standardgroup.

C—Represents the difference in paw volume from baseline in the controlgroup. A higher value of percentage inhibition indicated less paw volumein the animals as compared to control animals and more anti-inflammatoryactivity whereas a negative percentage inhibition indicated that testmaterial did not showing anti-inflammatory effect because the animalshad more paw volume than control animals at the corresponding timepoints.

As seen in Table 1 (FIG. 1), at each time point the rats treated withregular turmeric extract (Group 3) did not show percentage inhibition ofinflammation. The rats treated with curcuminoid mixture having C:D:B in0.9:1:1.1 w/w ratio (Group 5) showed slightly better percentageinhibition of inflammation than Group 3. Rats treated with regularturmeric extract blended with essential oil of turmeric having 45%Ar-turmerone in 10:1 ratio (Group 4) showed better percentage inhibitionof inflammation than Group 3 or Group 5 and almost comparable to ratstreated with Diclofenac (Group 2). Rats treated with curcuminoid mixturehaving C:D:B blended with essential oil of turmeric having 45%Ar-turmerone in 10:1 (Group 6) and 12:1 (Group 7) ratios showed betterpercentage inhibition of inflammation than rats treated with regularturmeric extract (Group 3) or regular turmeric extract blended withessential oil of turmeric having 45% Ar-turmerone in 10:1 ratio (Group4) or curcuminoid mixture alone having C:D:B in 0.9:1:1.1 w/w ratio(Group 5) or diclofenac (Group 2).

Example 9 Bio Availability of Curcumin, Demethoxycurcumin andBisdemethoxycurcumin in Rabbits

Rabbits weighing 2-2.5 Kg were used for the study. Animals were dividedinto 8 groups and 4 animals were used for each group. The animals weredivided as shown in Table 2. The dosage administered was 60 mgcurcuminoids/Kg body weight of the rabbit.

TABLE 2 Segregation of rabbits for experimental study. Group 1 Control(Tween 80) Dose: 60 mg of Tween 80/Kg body weight. Group 2 Regularturmeric extract prepared as per Example 1. Dose: Regular turmericextract (equivalent to 60 mg curcuminoids) solubilized in Tween 80/Kgbody weight. Group 3 Regular turmeric extract blended with EOT having45% Ar- turmerone in 10:1 ratio prepared as per Example 3. Dose: Regularturmeric extract blended with EOT having 45% Ar-turmerone in 10:1 ratio(equivalent to 60 mg curcuminoids) solubilized in Tween 80/Kg bodyweight. Group 4 Essential oil of turmeric having 45% Ar-turmeroneprepared as per Example 2. Dose: 60 mg of Essential oil of turmerichaving 45% Ar- turmerone solubilized in Tween 80/Kg body weight. Group 5Curcuminoid mixture having C:D:B in 0.9:1:1.1 w/w ratio prepared as perExample 4. Dose: 60 mg of Curcuminoid mixture having C:D:B in 0.9:1:1.1w/w ratio solubilized in Tween 80/Kg body weight. Group 6 Curcuminoidmixture having C:D:B in 0.9:1:1.1 w/w ratio blended with EOT having 45%Ar-turmerone in 10:1 ratio prepared as per Example 5. Dose: 60 mg of theformulation (Curcuminoid mixture having C:D:B in 0.9:1:1.1 w/w ratioblended with EOT having 45% Ar-turmerone in 10:1 ratio) solubilized inTween 80/Kg body weight. Group 7 Curcuminoid mixture having C:D:B in0.9:1:1.1 w/w ratio blended with EOT having 45% Ar-turmerone in 12:1ratio prepared as per Example 6. Dose: 60 mg of the formulation(Curcuminoid mixture having C:D:B in 0.9:1:1.1 w/w ratio blended withEOT having 45% Ar-turmerone in 12:1 ratio) in solubilized Tween 80/Kgbody weight. Group 8 Curcuminoid mixture having C:D:B in 0.9:1: 1.1 w/wratio blended with EOT having 45% Ar-turmerone in 1:10 ratio prepared asper Example 7. Dose: 60 mg of the formulation (Curcuminoid mixturehaving C:D:B in 0.9:1:1.1 w/w ratio blended with EOT having 45%Ar-turmerone in 1:10 ratio) solubilized in Tween 80/Kg body weight.

Group 1 consisted of animals which were given vehicle, Tween 80.

Group 2 animals were given regular turmeric extract (equivalent to 60mg/kg curcuminoids dose) (curcumin control) of which Curcumin 46.11 mg,Demethoxycurcumin 10.23 mg and Bisdemethoxycurcumin 3.66 mg solubilizedin Tween 80.

To Group 3 animals, regular turmeric extract blended with essential oilof turmeric having 45% Ar-turmerone in 10:1 ratio (Curcumin 41.92 mg,Demethoxycurcumin 9.3 mg and Bisdemethoxycurcumin 3.32 mg) solubilizedin Tween 80 and fed at dose equivalent to 60 mg/kg curcuminoids.

To Group 4 animals, essential oil of turmeric having 45% Ar-turmerone(60 mg/kg) in Tween 80 was given.

Curcuminoid mixture (60 mg/kg) having C:D:B in 0.9:1:1.1 w/w ratio withCurcumin 18 mg, Demethoxycurcumin 20 mg and Bisdemethoxycurcumin 22 mgwas solubilized in Tween 80 and fed to Group 5 animals.

To Group 6 animals, curcuminoid mixture having C:D:B in 0.9:1:1.1 w/wratio blended with essential oil of turmeric having 45% Ar-turmerone in10:1 ratio (Final formulation contain Curcumin 16.36 mg,Demethoxycurcumin 18.18 mg and Bisdemethoxycurcumin 20 mg and essentialoil of turmeric with 45% Ar-turmerone) solubilized in Tween 80 and fedat 60 mg/kg curcuminoid dose.

To group 7 animals, curcuminoid mixture having C:D:B in 0.9:1:1.1 w/wratio blended with essential oil of turmeric in 12:1 ratio (Finalformulation contain Curcumin 16.62 mg, Demethoxycurcumin 18.46 mg,Bisdemethoxycurcumin 20.30 mg and essential oil of turmeric with 45%Ar-turmerone.) solubilized in Tween 80 was fed at 60 mg/kg ofcurcuminoids.

To Group 8 animals, curcuminoid mixture blended with essential oil ofturmeric solubilized in Tween 80 was fed at 60 mg/kg curcuminoids. Thecurcuminoid mixture had a 0.9:1:1.1 w/w ratio ofcurcumin:demethoxycurcumin:bisdemethoxycurcumin. The essential oil ofturmeric had 45% Ar-turmerone. The ratio of curcuminoidmixture:essential oil of turmeric was 1:10 (Final formulation containCurcumin 1.64 mg, Demethoxycurcumin 1.82 mg and Bisdemethoxycurcumin 2mg).

The study drugs were given by oral route. Two hours post drug, blood wascollected from the ear vein of each rabbit for HPLC analysis of amountof curcumin, demethoxycurcumin and bisdemethoxycurcumin. The blood wasextracted exhaustively with ethyl acetate to recover the abovecomponents. The ethyl acetate extract was analyzed by HPLC on a RP-C18column (250×4.5 mm) using tetrahydrofuran (THF) as solvent and UVdetection at 420 nm. The eluent flow rate was 1 ml/min. Table 3 providesthe amount of curcumin, demethoxycurcumin and bisdemethoxycurcumin innanograms per gram of blood in rabbits by administering differentextracts.

TABLE 3 Bio availability of curcumin, demethoxycurcumin andbisdemethoxycurcumin in rabbits by administering different extracts.Demethoxy Bisdemethoxy Curcumin curcumin curcumin content in content incontent in Groups Treatment ng/g ng/g ng/g Group 1 Control (Tween 80) NDND ND Group 2 Regular turmeric 12.3 ± 1.9 ND ND extract Group 3 Regularturmeric 45.65 ± 2.3  ND ND extract blended with EOT having 45% Ar-t in10:1 ratio Group 4 Essential oil of ND ND ND turmeric having 45%Ar-turmerone Group 5 Curcuminoid mixture  6.3 ± 0.31  1.5 ± 0.44   2.9 ±0.89 having C:D:B in 0.9:1:1.1 w/w ratio Group 6 Curcuminoid mixture91.6 ± 2.3 98.5 ± 3.1 105.8 ± 5.2 having C:D:B in 0.9:1:1.1 w/w ratioblended with EOT having 45% Ar-t in 10:1 ratio Group 7 Curcuminoidmixture 101.5 ± 2.3  107.4 ± 2.5  112.9 ± 3.4 having C:D:B in 0.9:1:1.1w/w ratio blended with EOT having 45% Ar-turmerone in 12:1 ratio Group 8Curcuminoid mixture  1.3 ± 0.21 ND ND having C:D:B in 0.9:1:1.1 w/wratio blended with EOT having 45% Ar-turmerone in 1:10 ratio

The results as shown in Table 3 indicate that curcumin,demethoxycurcumin and bisdemethoxy curcumin was detected in group 5,group 6 and group 7. The level of curcuminoids in the blood was higherin rabbits fed with curcuminoid mixture blended with essential oil ofturmeric having 45% Ar-turmerone in 10:1 or in 12:1 ratios (Group 6 and7) compared to rabbits fed with curcuminoid mixture (Group 5) orcurcuminoid mixture blended with essential oil of turmeric having 45%Ar-turmerone in 1:10 ratio (Group 8). In group 5 rabbits fed withcurcuminoid mixture having C:D:B in 0.9:1:1.1, the level of curcumin,demethoxy curcumin and bisdemethoxycurcumin was found to be 6.3±0.31,1.5±0.44 and 2.9±0.89 respectively. Where as in Group 6 animals fed withcurcuminoid mixture blended with essential oil of turmeric having 45%Ar-turmerone in 10:1, the level of curcumin, demethoxycurcumin andbisdemethoxy curcumin in blood was 91.6±2.3, 98.5±3.1, 105.8±5.2 ng/grespectively. In group 7 animals fed with curcuminoid mixture blendedwith essential oil of turmeric having 45% Ar-turmerone in 12:1 has shownslightly higher curcuminoid level (curcumin, demethoxycurcumin andbisdemethoxy curcumin in blood was 101.5±2.3, 107.4±2.5, 112.9±3.4 ng/grespectively) than the rabbits fed with curcuminoid mixture blended withessential oil of turmeric having 45% Ar-turmerone in 10:1 ratio. Theseresults indicate that combining essential oil of turmeric withcurcuminoid mixture having C:D:B in 0.9:1:1.1 ratio increases the bloodconcentration of curcuminoids to higher level. Animals fed with regularturmeric extract (Group 2) shows only low detection of curcumin(12.3±1.9 ng/gm) in blood. Demethoxycurcumin and bisdemethoxy curcuminwas not detected in rabbits fed with regular turmeric extract.

Animals fed with regular turmeric extract blended with essential oil ofturmeric having 45% Ar-turmerone in 10:1 ratio (Group 3) shows detectionof curcumin (45.65±2.3 ng/gm) in blood. The curcuminoid content in theblood in animals fed with essential oil of turmeric having 45%Ar-turmerone (Group 4) was not detectable.

Example 10 Tissue Distribution Study in Rabbits

Rabbits weighing 2-2.5 Kg were used for the study. Animals were dividedinto 6 groups. The animals were divided as shown in Table 4. The dosageadministered was 60 mg curcuminoids/Kg body weight of the rabbit.

TABLE 4 Segregation of rabbits for experimental study. Group 1 Control(Tween 80) Dose: 60 mg of Tween 80/Kg body weight. Group 2 Regularturmeric extract prepared as per Example 1. Dose: Regular turmericextract (equivalent to 60 mg curcuminoids) solubilized in Tween 80/Kgbody weight. Group 3 Regular turmeric extract blended with EOT having45% Ar- turmerone in 10:1 ratio prepared as per Example 3. Dose: Regularturmeric extract blended with EOT having 45% Ar-turmerone in 10:1 ratio(equivalent to 60 mg curcuminoids) solubilized in Tween 80/Kg bodyweight. Group 4 Curcuminoid mixture having C:D:B in 0.9:1:1.1 w/w ratioalone prepared as per Example 4. Dose: 60 mg of Curcuminoid mixturehaving C:D:B in 0.9:1:1.1 w/w ratio solubilized in Tween 80/Kg bodyweight. Group 5 Curcuminoid mixture having C:D:B in 0.9:1:1.1 w/w ratioblended with EOT having 45% Ar-turmerone in 10:1 ratio prepared as perExample 5. Dose: 60 mg of the formulation (Curcuminoid mixture havingC:D:B in 0.9:1:1.1 w/w ratio blended with EOT having 45% Ar-turmerone in10:1 ratio) solubilized in Tween 80/Kg body weight. Group 6 Curcuminoidmixture having C:D:B in 0.9:1:1.1 w/w ratio blended with EOT having 45%Ar-turmerone in 12:1 ratio prepared as per Example 6. Dose: 60 mg of theformulation (Curcuminoid mixture having C:D:B in 0.9:1:1.1 w/w ratioblended with EOT having 45% Ar-turmerone in 12:1 ratio) solubilized inTween 80/Kg body weight.

Group 1 consisted of animal which were given vehicle, Tween 80.

Group 2 animals were given Regular turmeric extract of which Curcumin46.11 mg, Demethoxycurcumin 10.23 mg and Bisdemethoxy-curcumin 3.66 mg(equivalent to 60 mg curcuminoids) solubilized in Tween 80.

Group 3 animals were fed Regular turmeric extract blended with essentialoil of turmeric having 45% Ar-turmerone in 10:1 ratio (Curcumin 41.92mg, Demethoxycurcumin 9.3 mg and Bisdemethoxycurcumin 3.32 mg)solubilized in Tween 80.

Group 4 animals were orally administered with Curcuminoid mixture havingC:D:B in 0.9:1:1.1 w/w ratio with Curcumin 18 mg, Demethoxycurcumin 20mg and Bisdemethoxycurcumin 22 mg solubilized in Tween 80.

To Group 5 animals, Curcuminoid mixture having C:D:B in 0.9:1:1.1 w/wratio blended with essential oil of turmeric having 45% Ar-turmerone in10:1 ratio (Final formulation contain Curcumin 16.36 mg,Demethoxycurcumin 18.18 mg, Bisdemethoxycurcumin 20 mg and essential oilof turmeric with 45% Ar-turmerone) solubilized in Tween 80 was fed.

To Group 6 animals, Curcuminoid mixture having C:D:B in 0.9:1:1.1 w/wratio blended with essential oil of turmeric having 45% Ar-turmerone in12:1 ratio (Final formulation contain Curcumin 16.62 mg,Demethoxycurcumin 18.46 mg, Bisdemethoxycurcumin 20.30 mg and essentialoil of turmeric with 45% Ar-turmerone) solubilized in Tween 80 was fed.

The study drugs were given by oral route. Two hours post drug rabbitswere sacrificed by cervical dislocation and dissected to obtain varioustissues (Heart, Kidney, Brain, Liver, Pancreas, Lungs, Intestine,Stomach and Skin). Each tissue was weighed and stored in vial at −20° C.until analysis.

Tissues were homogenized and extracted with chloroform-methanol (2:1).The homogenate was filtered through a whatman filter paper and thefiltrate collected and mixed thoroughly with 0.2 vol of 0.9% NaClsolution and centrifuged. The upper layer was siphoned off. The lowerphase was evaporated to dryness under a stream of nitrogen at 40-45° C.in a “Turbo Vap” Concentration Work Station (Caliper Life Sciences,USA).

Dried samples of tissues were analyzed on HPLC. The dried samples weredissolved in 2 ml of acetone using a vortex mixer and analyzed by HPLCin a Shimadzu LC 20AD Liquid Chromatograph system with SPD-M20A UVdetector in isocratic mode. The column used was C18 ODS Phenomenox(250×4.6 mm, 5μ particle size) using 40% tetrahydrofuran (THF), 60%water containing 1% citric acid (P^(H) adjusted to 3 with concentratedKOH solution) as solvent system and UV detection at 420 nm. The eluentflow rate was 1 ml/min.

Group 1 (Control group) Curcumin Sl No:- Tissues (ng/g) DMC BDMC 1 LungsND ND ND 2 Heart ND ND ND 3 Kidney ND ND ND 4 Brain ND ND ND 5 Liver NDND ND 6 Pancreas ND ND ND 7 Stomach ND ND ND 8 Intestine ND ND ND 9 SkinND ND ND

Group 2 - Regular turmeric extract Curcumin DMC BDMC Sl No:- Tissuesng/g ng/g ng/g 1 Lungs 4.2 ND ND 2 Heart 17.5 ND ND 3 Kidney ND ND ND 4Brain 5.3 ND ND 5 Liver 10 ND ND 6 Pancreas 13.4 ND ND 7 Stomach 856 NDND 8 Intestine 618 ND ND 9 Skin 2.1 ND ND

Group 3 - Regular turmeric extract blended with EOT having 45%Ar-turmerone in 10:1 ratio Curcumin DMC BDMC Sl No:- Tissues ng/g ng/gng/g 1 Lungs 22.1 ND ND 2 Heart 235.6 ND ND 3 Kidney 21.5 ND ND 4 Brain19.6 ND ND 5 Liver 87.5 ND ND 6 Pancreas 223.1 ND ND 7 Stomach 7456 NDND 8 Intestine 3562 ND ND 9 Skin 12.4 ND ND

Group 4 - Curcuminoid mixture having C:D:B in 0.9:1:1.1 w/w ratioCurcumin DMC BDMC Sl No:- Tissues ng/g ng/g ng/g 1 Lungs 5.8 ND ND 2Heart 16.9 1.3 ND 3 Kidney 1.2 ND ND 4 Brain 6.1 1.1 ND 5 Liver 25 3.4ND 6 Pancreas 14.3 2.5 ND 7 Stomach 848 122    ND 8 Intestine 612 180   ND 9 Skin 1.9 1.1 ND

Group 5 - Curcuminoid mixture having C:D:B in 0.9:1:1.1 w/w ratioblended with EOT having 45% Ar-turmerone in 10:1 ratio Curcumin DMC BDMCSl No:- Tissues ng/g ng/g ng/g 1 Lungs 32.1 13.5 9.8 2 Heart 356.77 292243 3 Kidney 29.95 7.2 12.6 4 Brain 29.76 11.7 8.2 5 Liver 122.15 38.2622.14 6 Pancreas 357 232 114.5 7 Stomach 12135 10356 9845 8 Intestine5634 3562 1123 9 Skin 16.2 9.5 7.2

Group 6 - Curcuminoid mixture having C:D:B in 0.9:1:1.1 w/w ratioblended with EOT having 45% Ar-turmerone in 12:1 ratio Curcumin DMC BDMCSl No:- Tissues ng/g ng/g ng/g 1 Lungs 40.2 14.8 16.2 2 Heart 402.1 273251 3 Kidney 42.8 36.8 11.5 4 Brain 38.4 15.8 13.4 5 Liver 154.1 49.728.4 6 Pancreas 389.7 251.5 122.6 7 Stomach 15987 13842 10245 8Intestine 7562 4563 1562 9 Skin 19.5 13.6 10.3

Pancreas, Heart and Liver showed a better absorption at 2 hrs afterdosing curcuminoid mixture having C:D:B in 0.9:1:1.1 ratio blended withessential oil of turmeric having 45% Ar-turmerone in 10:1 or in 12:1ratios. Kidney, lungs, skin and brain showed moderate amounts ofcurcumin, demethoxycurcumin and bisdemethoxycurcumin after dosingcurcuminoid mixture having C:D:B in 0.9:1:1.1 ratio blended withessential oil of turmeric having 45% Ar-turmerone in 10:1 or in 12:1ratios.

In Group 2 and 3 animals after consuming regular turmeric extract andregular turmeric extract blended with essential oil of turmeric having45% Ar-turmerone, all the tissues showed absorption of curcumin. In thecontrol group (Group 1) there was no absorption of curcuminoids in thetissues. All other tissues like Spleen, Uterus, Seminal Vesicle, Testisand Prostate also showed significant absorption of curcumin,demethoxycurcumin and bisdemethoxycurcumin after dosing with curcuminoidmixture having C:D:B in 0.9:1:1.1 ratio blended with essential oil ofturmeric having 45% Ar-turmerone in 10:1 or in 12:1 ratios.

Example 11 Bioavailability of Curcumin, Demethoxycurcumin andBisdemethoxycurcumin in Humans

Nine healthy human volunteers aged between 25 and 45 years of age wereselected for the study. They were given capsules containing Regularturmeric extract (equivalent to 2000 mg curcuminoids) prepared as perExample 1 (Curcumin 1536.84 mg, Demethoxycurcumin 341.05 mg andBisdemethoxycurcumin 122.11 mg). Blood was drawn from the subjects atbaseline, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8 and 10 hours post drug. Thesame subjects after a washout period of one week were given doseequivalent to 2000 mg curcuminoids having regular turmeric extractblended with essential oil of turmeric having 45% Ar-turmerone in 10:1ratio prepared as per Example 3 (Curcumin 1397.1 mg, Demethoxycurcumin310.05 mg and Bisdemethoxycurcumin 111.01 mg). Blood was drawn from thesubjects at baseline, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8 and 10 hours postdrug.

The above protocol was repeated with the following formulation also:

-   -   (1) Capsule having 500 mg of curcuminoid mixture having C:D:B in        0.9:1:1.1 w/w ratio prepared as per Example 4 (Curcumin 600 mg,        Demethoxycurcumin 666.67 mg and Bis-demethoxycurcumin 733.33 mg)        was administered at a dosage of 4×500 mg (equivalent to 2000 mg        curcuminoids) of composition to the human subject.    -   (2) Capsule having 500 mg of curcuminoid mixture in 0.9:1:1.1        w/w ratio (C:D:B) blended with EOT having 45% Ar-turmerone in        10:1 ratio prepared as per Example 5 (Final formulation contain        Curcumin 545.45 mg, Demethoxycurcumin 606.06 mg,        Bisdemethoxycurcumin 666.6 mg and essential oil of turmeric with        45% Ar-turmerone) was administered at a dosage of 4×500 mg        (equivalent to 2000 mg curcuminoids) of composition to the human        subject.    -   (3) Capsule having 500 mg of curcuminoid mixture in 0.9:1:1.1        w/w ratio (C:D:B) blended with EOT having 45% Ar-turmerone in        12:1 ratio prepared as per Example 6 (Final formulation contain        Curcumin 553.8 mg, Demethoxycurcumin 615.38 mg,        Bisdemethoxycurcumin 676.92 mg and essential oil of turmeric        with 45% Ar-turmerone) was administered at a dosage of 4×500 mg        (equivalent to 2000 mg curcuminoids) of composition to the human        subject.

The blood was extracted exhaustively with ethyl acetate to recover theCurcumin, Demethoxycurcumin and Bisdemethoxycurcumin components. Theethyl acetate extract was analyzed by HPLC on a RP-C18 column (250×4.5mm) using tetrahydrofuran (THF) as solvent and UV detection at 420 nm.The eluent flow rate was 1 ml/min. Table 5 (FIG. 2) provides the amountof curcumin, demethoxycurcumin and bisdemethoxycurcumin in nanograms pergram of blood for the subjects by administering different extracts.

Results showed that curcumin, demethoxycurcumin and bisdemethoxycurcuminwere detected in groups treated with curcuminoid mixture having C:D:B in0.9:1:1.1 w/w ratio alone and curcuminoid mixture having C:D:B in0.9:1:1.1 w/w ratio blended with essential oil of turmeric having 45%Ar-turmerone in 10:1 and 12:1 ratios. The curcuminoids was detected forup to 10 hr in subjects treated with curcuminoid mixture having C:D:B in0.9:1:1.1 w/w ratio blended with essential oil of turmeric having 45%Ar-turmerone in 10:1 and 12:1 ratios. Following administration ofcapsules having a 10:1 weight ratio of curcuminoid mixture having C:D:Bin 0.9:1:1.1 w/w ratio blended with essential oil of turmeric having 45%Ar-turmerone, the peak absorption of curcumin, demethoxycurcumin andbisdemethoxycurcumin occurred at 3 hr. The maximum absorption forcurcumin was 102.6 ng/gm, for demethoxycurcumin 120.2 ng/gm and forbisdemethoxycurcumin 140.7 ng/gm in the group fed by 10:1 weight ratioof curcuminoid mixture having C:D:B in 0.9:1:1.1 w/w ratio blended withessential oil of turmeric having 45% Ar-turmerone. The subjects treatedwith curcuminoids having C:D:B in 0.9:1:1.1 w/w ratio blended withessential oil of turmeric having 45% Ar-turmerone in 12:1 ratio showedslightly higher curcumin, demethoxycurcumin and bisdemethoxycurcuminlevel at 3 hr. The maximum absorption for curcumin was 118.1 ng/gm, fordemethoxycurcumin 125.1 ng/gm and for bisdemethoxycurcumin 152.5 ng/gmin the group fed by 12:1 weight ratio of curcuminoid mixture havingC:D:B in 0.9:1:1.1 w/w ratio blended with essential oil of turmerichaving 45% Ar-turmerone.

Subjects treated with curcuminoid mixture having C:D:B in 0.9:1:1.1 w/wratio showed low detection of curcumin (13.9 ng/gm), demethoxycurcumin(3.6 ng/gm) and bisdemethoxycurcumin (3.5 ng/gm).

Subjects treated with curcuminoid mixture having C:D:B in 0.9:1:1.1 w/wratio blended with essential oil of turmeric having 45% Ar-turmerone in10:1 and 12:1 ratios has shown higher detection of curcuminoids even at10 hrs post drug. Whereas curcuminoid mixture having C:D:B in 0.9:1:1.1w/w ratio alone gave a low detection of curcuminoids at 6 hrs.

Demethoxycurcumin and bisdemethoxycurcumin was not detected in thesubjects treated with regular turmeric extract and regular turmericextract blended with essential oil of turmeric. The subjects treatedwith regular turmeric extract blended with essential oil of turmerichaving 45% Ar-turmerone in 10:1 ratio showed detection of curcumin onlyin the blood and Cmax was found to be 93.56 ng/gm. Curcumin was detectedup to 8 hr in the blood.

Subjects treated with regular turmeric extract showed only low detectionof curcumin (15.3 ng/gm) in blood.

Other modifications and variations to the invention will be apparent tothose skilled in the art from the foregoing disclosure and teachings.Thus, while only certain embodiments of the invention have beenspecifically described herein, it will be apparent that numerousmodifications may be made thereto without departing from the spirit andscope of the invention.

What is claimed is:
 1. A method of decreasing inflammation comprisingadministering a composition for enhanced bioavailability of curcumin,demethoxycurcumin and bisdemethoxycurcumin, the composition comprising acurcuminoid mixture and essential oil of turmeric, wherein thecurcuminoid mixture comprises curcumin, demethoxycurcumin andbisdemethoxycurcumin in a weight ratio ofcurcumin:demethoxycurcumin:bisdemethoxycurcumin of about 0.8:1:1 toabout 1:1.1:1.2, wherein the essential oil of turmeric comprises about45% Ar-turmerone, and, wherein a weight ratio of the curcuminoid mixtureto essential oil of turmeric ranges from about 1:3 to about 99:1.
 2. Themethod of decreasing inflammation of claim 1, wherein the weight ratioof the curcuminoid mixture to essential oil of turmeric is about 12:1.3. The method of decreasing inflammation of claim 1, wherein the weightratio of the curcuminoid mixture to essential oil of turmeric is about10:1.
 4. The method of decreasing inflammation of claim 1, wherein adecrease in inflammation is observed from about 30 mins to about 24hours.
 5. A method of enhancing bioavailability of curcumin,demethoxycurcumin and bisdemethoxycurcumin comprising administering acomposition for enhanced bioavailability of curcumin, demethoxycurcuminand bisdemethoxycurcumin, the composition comprising a curcuminoidmixture and essential oil of turmeric, wherein the curcuminoid mixturecomprises curcumin, demethoxycurcumin and bisdemethoxycurcumin in aweight ratio of curcumin:demethoxycurcumin:bisdemethoxycurcumin of about0.8:1:1 to about 1:1.1:1.2, and the essential oil of turmeric comprisesabout 45% Ar-turmerone, and, wherein a weight ratio of the curcuminoidmixture to essential oil of turmeric ranges from about 1:3 to about99:1.
 6. The method of enhancing bioavailability of curcumin,dimethoxycurcumin and bisdemethoxycurcumin of claim 5, wherein theweight ratio of the curcuminoid mixture to essential oil of turmeric isabout 12:1.
 7. The method of enhancing bioavailability of curcumin,dimethoxycurcumin and bisdemethoxycurcumin of claim 5, wherein theweight ratio of the curcuminoid mixture to essential oil of turmeric isabout 10:1.
 8. The method of enhancing bioavailability of curcumin,dimethoxycurcumin and bisdemethoxycurcumin of claim 5, wherein anenhancement of bioavailability of curcumin, dimethoxycurcumin andbisdemethoxycurcumin is observed in a tissue, and wherein the tissue isselected from the group consisting of lungs, heart, kidney, brain,liver, pancreas, stomach, intestine and skin.